aem accepts, published online ahead of print on 5 december ...€¦ · 02/12/2014 · 5 83...

1

Running title: Genome-wide analysis of S. maltophilia biofilm genes 1

2

Genome-Wide Identification of Genes Necessary for Biofilm Formation of 3

Nosocomial Pathogen Stenotrophomonas maltophilia Reveals Orphan 4

Response Regulator FsnR is a Critical Modulator 5

6

Xiu-Min Kang1,2§

, Fang-Fang Wang1§

, Huan Zhang1,3

, Qi Zhang

2#, Wei Qian

1# 7

8

1 State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of 9

Sciences, Beijing 100101, China. 2 School of Chemical Engineering & Environment, Beijing 10

Institute of Technology, Beijing 100081, China. 3College of Life Sciences, University of 11

Chinese Academy of Sciences, Beijing 100049, China. 12

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§ These authors contributed equally to this work 14

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#Corresponding authors. Wei Qian, Tel: +86-10-64806063; Fax: +86-10-64858245; Email: 16

[email protected]. Qi Zhang, Tel: 86-10-68918012; Email: [email protected] 17

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Keywords: Stenotrophomonas maltophilia, biofilm, transposon mutagenesis, response 19

regulator, flagella assembly 20

AEM Accepts, published online ahead of print on 5 December 2014Appl. Environ. Microbiol. doi:10.1128/AEM.03408-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Abstract 21

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Stenotrophomonas maltophilia is a gram-negative, bacterial pathogen of increasing 23

concern to human health. Most clinical isolates of S. maltophilia efficiently form biofilms on 24

biotic and abiotic surfaces, making this bacterium resistant to a number of antibiotic 25

treatments and hard to be eliminated. To date, very few studies have investigated the 26

molecular and regulatory mechanisms responsible for S. maltophilia biofilm formation. Here 27

we constructed a random transposon insertion mutant library of S. maltophilia ATCC 13637 28

and screened 14,028 clones. A total of 46 non-redundant genes were identified. Mutants of 29

these genes exhibited marked changes in biofilm formation; suggesting multiple physiological 30

pathways, including extracellular polysaccharide production, purine synthesis, transportation, 31

peptide and lipid synthesis, were involved in bacterial cell aggregation. Of these genes, 20 32

putatively contributed to flagella biosynthesis, indicating a critical role for cell motility in S. 33

maltophilia biofilm formation. Genetic and biochemical evidence demonstrated an orphan 34

response regulator, FsnR, activated transcription of at least two flagella-associated operons by 35

directly binding to their promoters. The regulatory protein plays a fundamental role in 36

controlling flagella assembly, cell motility and biofilm formation. These results provide a 37

genetic basis to systematically study biofilm formation of S. maltophilia. 38


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INTRODUCTION 39

40

Stenotrophomonas maltophilia (formerly Pseudomonas maltophilia or Xanthomonas 41

maltophilia) are aerobic, gram-negative bacteria belonging to the family Xanthomonadaceae 42

of the γ-Proteobacteria. S. maltophilia are ubiquitous across a diverse range of ecological 43

niches, including foods, soil, plant roots and stems, and aqueous environments. In clinical 44

settings, S. maltophilia is an opportunistic, nosocomial pathogen, predominantly colonizing 45

the skin, respiratory tract, urinary catheters, and breathing tubes such as endotracheal tubes (1, 46

2). Infection generally results in pneumonia, bacteremia, urinary tract infection, and 47

meningitis (3, 4). Over the past three decades, S. maltophilia has become an increasing threat 48

to public health, in particular, immunosuppressed or immunocompetent patients in intensive 49

care units (ICU) undergoing prolonged mechanical ventilation, tracheostomy or 50

broad-spectrum antibiotic therapy (5). The reported mortality rates for S. maltophilia infected 51

patients range between 23% and 77% (4, 6). Treatment of S. maltophilia infection has proven 52

increasingly difficult because of the bacteria’s intrinsic multidrug resistance. S. maltophilia is 53

resistant to treatment with most aminoglycosides, quinolones, β-lactams, and β-lactamase 54

inhibitors (7). Worryingly, research has shown susceptibility of bacterial isolates to the 55

recommended drug for S. maltophilia infection, trimethoprim-sulfamethoxazole, has 56

decreased from >98% to 30–40% for within a decade (8). Therefore, the development of more 57

effective antibiotic agents targeting critical processes of S. maltophilia pathogenesis, 58

nonessential to survival and with minimal selective pressure, will be a challenge faced by 59

researchers in the future. 60

Biofilm is slime-enclosed bacterial aggregates affording protection against antibiotics, 61


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host immune responses, and multiple stresses (9). Most clinical strains of S. maltophilia form 62

biofilms efficiently on various abiotic and biotic surfaces, including Teflon, plastic, glass, host 63

tissue, and in-dwelling intravascular devices (10, 11). Environmental factors, including 64

phosphate, pH, temperature, metal ions, and antibiotics were shown to have a profound effect 65

on S. maltophilia biofilm formation (3). To date, however, only a small number of genes or 66

biochemical cascades associated with S. maltophilia biofilm formation have been reported, 67

and these include polysaccharide synthesis genes rmlA, rmlC, and xanB (12), DSF-mediated 68

cell-cell communication (13), small RNA modulator Hfq (14), and ABC-type efflux pump 69

MacABC (15). Hence, it is critical to undertake a systematic approach when elucidating the 70

molecular and regulatory properties of S. maltophilia biofilm development. 71

In this work we constructed a large scale S. maltophilia transposon insertional mutant 72

library which was used to screen for S. maltophilia mutants with altered biofilm forming 73

ability (16). From 14,208 clones we identified 46 genes belonging to mutants exhibiting 74

remarkable changes in biofilm development. Almost half of these genes (20 genes, 43.5%) 75

were involved in bacterial flagella or pili biosynthesis, which suggested cell motility play a 76

critical role in S. maltophilia biofilm development. We confirmed that FsnR, an orphan 77

response regulator of the two-component signal transduction system, directly binds to the 78

promoter region of two gene clusters and activates their transcription. These results provide 79

genetic information to further investigate the molecular process of S. maltophilia biofilm 80

formation. 81

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MATERIALS AND METHODS 83

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Bacterial strains, plasmids and culture conditions. The bacterial strains and plasmids used 85

in the present study are described in Table 1. Escherichia coli DH5α was used as the host in 86

molecular cloning procedures, and routinely cultured at 37°C in Luria-Bertani (LB) medium. 87

S. maltophilia ATCC 13637, obtained from stocks of CGMCC, was cultured at 28°C in NYG 88

medium (5 g/L tryptone, 3 g/L yeast extract, 20 g/L glycerol; pH 7.0). Competent cells of S. 89

maltophilia were prepared by culturing in 210 medium (4 g/L yeast extract, 8 g/L casein 90

enzymatic hydrolysate, 5 g/L sucrose, 3 g/L K2HPO4, 0.3 g/L MgSO4.7H2O; pH 7.0) and 91

washed in ice-cold glycerol (10%). Antibiotics were used at the following concentrations: 92

ampicillin, 100 μg/ml; kanamycin, 50 μg/ml; spectinomycin, 100 μg/ml; and streptomycin, 93

200 μg/ml. Electroporation conditions for the transformation of both S. maltophilia and E. 94

coli were set at 18 kVcm−1

, 25 μF and 200 Ω and conducted in a Bio-Rad Pulser XCell™ 95

Electroporation System (Hercules, USA). 96

97

Construction of insertional mutant library and genetic manipulation of bacterial strains. 98

The S. maltophilia transposon insertional mutant library was constructed using the EZ::TN 99

<KAN-2> Tnp transposome kit (Epicentre, USA), as described previously and in accordance 100

with the manufacturer’s protocol (17, 18). A total of 14,208 kanamycin resistant transformants 101

were selected and stored in 37 384-well plates at −80°C until use. Southern blot hybridization 102

was used to determine transgene copy number. In brief, total DNA was extracted from 103

bacterial strains, the electrophoresed fragments transferred to a Hybond N+ membrane, and 104

hybridized with a [α-32

P]-dCTP labelled PCR probe (Prime-A-Gene, Promega, USA). PCR 105


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primers used to amplify the kanamycin resistant sequence and used as DNA probes are listed 106

in Table 2. 107

General molecular cloning procedures, including PCR, DNA extraction and ligation, 108

restriction enzyme digestions, and Southern blotting were conducted as per the methods of 109

Sambrook (19). All primers used for mutagenesis, protein expression and RT-PCR are listed 110

in Table 2. The in-frame deletion mutant of fsnR was constructed by homologous, 111

double-crossover recombination using the suicide vector pk18mobsacB (20). Correction of 112

the in-frame deletion mutant was verified by PCR and sequencing. A genetic complementary 113

strain was constructed as outlined below. EcoRI and HindIII restriction sites were introduced 114

into full-length fsnR, the gene sequence amplified by PCR, the amplicon ligated with pHM1 115

broad-host vectors, and electroporated into S. maltophilia strains. In this construct, fsnR is 116

under the control of a placZ promoter. 117

118

Biofilm assay and quantification. The crystal violet staining method for the quantification of 119

biofilm formation was adapted from a previously described method (21, 22). In brief, S. 120

maltophilia cultures were inoculated quantitatively into NYG medium within 96-well 121

polystyrene plates and incubated at 28°C for 8 hours without shaking. The optical density 122

(OD600) for each well was measured on a TECAN Infinite 200 PRO microplate reader. The 123

wells were washed rigorously with water prior to staining with 1% crystal violet solution for 124

20 min. The wells were washed, the crystal violet stain solubilized in absolute ethanol, and 125

biofilm formation measured at OD590 on a microplate reader. Each data point was averaged 126

from at least four replicates. 127

128

Swimming motility and transmission electron microscopy. For the swimming motility 129


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assay, bacterial strains were inoculated into 0.1% NYG agar with a toothpick, cultured for 24 130

hours at 28°C, and the diameters of the swimming zones measured. 131

Transmission electron microscopy was used to observe flagella morphology. Briefly, 132

bacterial suspensions prepared from cultures of S. maltophilia were placed directly on 133

glow-discharged carbon-coated grids, stained with 2.0% uranyl acetate for 2 min, washed 134

twice with water, and air dried. Bacterial flagella were then visualized using a JEDL 135

JEM-1400 transmission electron microscope (JEOL, USA). 136

137

RNA extraction and semi-quantitative RT-PCR. Total bacterial RNA was extracted using 138

TRIzol (Invitrogen, USA) according to the manufacturer’s instructions. The extracted RNA 139

was quantified with a NanoDrop spectrophotometer (Thermo Fisher, USA). Total RNA was 140

treated with DNA-free DNAse (Life Technologies, USA) to remove contaminating DNA. The 141

first strand of cDNA was synthesized using random primers (Promega, USA) and Superscript 142

III reverse transcriptase (Invitrogen, USA). Controls used in the semi-quantitative RT-PCR 143

assay are as follows: DNA templates of the WT strain were included as the positive control 144

for PCR, tmRNA amplification was the loading control for RT-PCR, and samples that lacked 145

reverse transcriptase during cDNA synthesis were included as negative controls to evaluate 146

potential DNA contamination. Primers used for the amplification of sample genes and tmRNA 147

genes are listed in Table 2. 148

149

Protein expression and electrophoretic mobility shift assay (EMSA). Recombinant 150

FsnR-His6 protein was expressed in E. coli BL21(DE3) cells transformed with a recombinant 151

pET30a (Novagen) expression vector. PCR primers used in the construction of the 152


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recombinant vector are listed in Table 2. FsnR-His6 protein was purified by Ni-NTA affinity 153

chromatography according to the manufacturer’s instructions (Novagen). The purified protein 154

was concentrated using Centricon YM-10 columns (Millipore, Austrilia), and the buffer 155

exchanged with storage buffer (50 mM Tris-HCl, 0.5 mM EDTA, 50 mM NaCl, 5% glycerol; 156

pH 8.0) for further use. 157

For the EMSA, promoter regions of the Smlt2303 and Smlt2318 operons were amplified 158

by PCR (Table 2 for primer sequences). The PCR products (299 bp and 267 bp, respectively) 159

were labeled with [α-32

P]-dCTP using the Prime-A-Gene Kit (Promega, USA), and free 160

[α-32

P]-dCTP removed by a ProbeQuant G-50 column (GE, USA). DNA probes (4 fmol) and 161

proteins (1 μg) were co-incubated in reaction buffer containing 10 mM Tris (pH 7.0), 50 mM 162

KCl, 1 mM DTT (pH 7.5), 2.5% glycerol, 5 μl MgCl2, 50 ng/μl poly(dI:dC), 0.05% NP-40, 163

and 10 mM EDTA for 20 minutes at room temperature. A 4 μl aliquot of DNA loading buffer 164

(0.25% bromophenol blue, 40% sucrose) was added to stop the reaction and the samples 165

loaded into a 5% native PAGE gel. Electrophoresis was performed at 120 V for about 40 min 166

using 0.5 × TBE buffer. The gel was placed in a zip-lock bag and phosphor imaging screens 167

used for signal detection. Unlabeled probes were used in the competitive experiments. 168


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RESULTS 169

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Construction and screening of the random insertion mutant library of S. maltophilia 171

S. maltophilia ATCC 13637 is a clinical strain isolated from the oropharyngeal region of 172

a patient with mouth cancer. It was chosen for use in the current study as it rapidly forms 173

biofilms within several hours. A random, Tn5 transposon insertion mutant library was 174

constructed with the EZ::TN transposome system (16). The library comprises 37 384-well 175

plates and 14,208 kanamycin resistant transformants. To evaluate the randomness and 176

proportion of single-copy insertions, total DNA from 29 randomly chosen clones was 177

extracted, digested with SmaI, and analyzed by Southern blotting. As shown in Figure 1A, 27 178

clones (93%) contained single-copy insertions, whilst two clones (7%, lanes 13 and 14) were 179

simultaneously inserted with two EZ::TN transposons. Additionally, six clones cultivated in 180

NYGB media in the absence of kanamycin for 20 generations maintained their resistance to 181

kanamycin, confirming the stability of the transposon inserts (Fig. S1 in supplementary 182

information). 183

The library was then screened for mutants with altered biofilm-forming capabilities using 184

the crystal violet staining assay. Bacterial growth was also quantified to eliminate potential 185

growth-associated contributions to changes in biofilm formation. The primary screening 186

round identified a total of 396 clones exhibiting changes in the quantity of biofilm on the 187

polypropylene surface (Fig. 1B). These clones were selected, propagated in 96-well plates, 188

and subjected to four additional rounds of screening by biofilm assay. Of the 396 clones 189

selected, 244 were identified as false-positive because their phenotypic change failed to be 190

stably replicated throughout the screening process. Additionally, 53 clones with poor or no 191


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growth were excluded from subsequent analyses because the decrease in biofilm formation 192

could be caused by auxotrophy or slow growth. Thermal asymmetric interlaced (TAIL)-PCR 193

was used to identify genomic sequences flanking the EZ::TN transposon insertion sites for 99 194

clones exhibiting decreased (80 clones) and increased (19 clones) biofilm formation. 195

196

Identification of genes responsible for biofilm formation emphasizes the role of flagella 197

biosynthesis 198

TAIL-PCR analysis successfully identified the transposon insertional sites in each of 199

these clones. A total of 46 non-redundant genes, 4 intergenic regions, and 2 undefined 200

insertion sites within a multicopy locus were identified (Fig. 2A and Table S1). Because the 201

genome of S. maltophilia ATCC 13637 has not been sequenced, the inserted genes were 202

mapped onto the complete genome of S. maltophilia reference strain K279a (1). The mutant 203

genes can be functionally classified into nine categories: 1) flagella and pili assembly; 2) 204

polysaccharide biosynthesis, e.g. xanA; 3) peptide and lipid synthesis; 4) purine biosynthesis, 205

e.g. purE, purK, guaA, and purC; 5) transmembrane transportation; 6) gene expression 206

regulation; 7) oxidoreduction; 8) transposable elements, and 9) unknown function (Fig. 2B 207

and Table S1). Overall, mutations identified in five genes were associated with an increase in 208

biofilm formation, whilst the remaining 41 genes showed deficits. Southern blotting analysis 209

revealed that all the above identified mutants contain a single-copy of transposon (Fig. S2). 210

Almost half of the genes identified (20 genes, 43.5%) were associated with flagella 211

structure and regulation; highly suggestive that the flagellum plays a prominent role in S. 212

maltophilia biofilm formation. As shown in Figure 3, these genes were organized into 10 213

putative operons in the genome of S. maltophilia reference strain K279a. These operons 214


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encode structural and assembly proteins associated with different stages of flagellar assembly. 215

FlhA, FliO, FliN, FliM, FliI, FliF, FlgI, FlgH, and FlgG are structural components of the basal 216

body. FliD is the flagellar filament capping protein, whilst FliK controls flagellar hook length 217

during assembly. FlhF is a putative GTPase that is often necessary for assembly and 218

placement of polar flagellar. FliJ acts as a chaperone, exporting flagellar proteins from the 219

cytoplasm into the periplasm. FlgL, FlgK, FlgJ, and FlgE are components of the flagellar 220

hook and assist in connecting the basal body to the filament. 221

It is noteworthy, that five regulatory genes were identified within or in the vicinity of 222

flagellar gene clusters. Smlt2270 (fliA) and Smlt2297 encode σ28

and σ54

, respectively. These 223

alternative sigma factors are involved in regulating flagellar gene transcription. Smlt0158 224

encodes a histidine kinase (NRII or GlnL) which may phosphorylate the putative cognate 225

response regulator Smlt0159 (NRI or GlnG). Smlt2324 encodes a PAS/PAC 226

domain-containing histidine kinase that is orthologous to RavS of Xanthomonas campestris 227

(23). In the vicinity of ravS, there was another histidine kinase gene (ravA, Smlt2322) and a 228

GGDEF/EAL domain-containing response regulator gene Smlt2323 (orthologue of ravR). 229

Additionally, a response regulator gene, Smlt2299, was also identified. Smlt2299 encodes a 230

putative protein product with 210 amino acid residues in length. The protein contains an 231

N-terminal receiver domain for accepting phosphoryl groups, and a C-terminal LuxR-type 232

DNA-binding domain. In the absence of a histidine kinase gene near Smlt2299, its product 233

Smlt2299 was considered an “orphan” response regulator. To our knowledge Smlt2299 and its 234

orthologues have not been reported previously. As this gene is not an ortholog of known 235

flagella-associated response regulators, such as FlgR of Campylobacter jejuni and 236

Helicobacter pylori, we proposed “FsnR” was a more suitable name for the flagella 237


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biosynthesis regulator Smlt2299. The regulatory role of FsnR was investigated further in the 238

current study. 239

240

FsnR controls biofilm development and flagella biosynthesis 241

Since fsnR is putatively located in an operon and transposon insertion may lead to polar 242

effect to complicate the genetic analysis, we constructed a non-polar, in-frame deletion mutant 243

of fsnR via homologous double cross-over recombination. This mutant strain was labeled 244

SM001 (ΔfsnR). To generate the complement gene mutation, the full-length sequence of fsnR 245

was amplified by PCR, cloned into the broad-host vector pHM1, and transformed into SM001, 246

giving rise to strain SM002 (ΔfsnR-fsnR). In this construct, fsnR was under the control of the 247

placZ promoter. As shown in Figures 4A and 4B, SM001 biofilm production had decreased 248

significantly to 27.5% of WT strain, whilst genetic complementation substantially restored the 249

bacteria’s biofilm forming ability (77.6% of WT strain). Similar growth curves were observed 250

for SM001 and WT strain, whilst SM002 grew comparatively slower (Fig. 4C). The disparity 251

in growth curves may be caused by overexpression of the fsnR gene for SM002. These results 252

demonstrated that mutations in the fsnR gene were responsible for deficiencies in bacterial 253

biofilm formation, and that the phenotypic change was not due to changes in growth rate. 254

Because fsnR resides in an operon with fliS, fliD, and two as yet functionally unknown 255

genes Smlt2300 and Smlt2301, we surmised Smlt2299 may be required for the regulation of 256

flagellar biosynthesis and bacterial motility. As such, swimming motility and flagella 257

morphology were investigated for each of the bacterial strains. The mutant SM001 was found 258

to be non-motile on semi-solid NYG agar plates containing 0.1% agar, whilst genetic 259

complementation partially restored the swimming ability of SM002 to 50.5% of WT strain 260


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(Fig. 5A). Transmission electron microscopy confirmed the presence of clearly visible polar, 261

single flagellum for most WT and SM002 cells, whilst SM001 cells lacked flagella (Fig. 5B). 262

These results demonstrate that the response regulator FsnR controls flagella biosynthesis and 263

cell motility. 264

265

FsnR directly binds to promoters of flagellar genes and activates their transcription 266

Semi-quantitative RT-PCR was used to establish whether FsnR is a critical transcription 267

factor in controlling flagellar gene expression. The transcription levels of 11 genes located in 268

the 5' region of operons associated with flagella assembly (Fig. 3) were measured. As shown 269

in Figure 6A, with the exception of Smlt0710 whose expression was at undetectable levels, 270

the quantity of mRNA for these representative genes was markedly reduced in the fsnR 271

mutant. In contrast, the transcriptional levels of all genes was either restored, or increased to 272

levels greater than for WT, for complementary strain SM002. This result suggests that FsnR is 273

a canonical, positive regulator, directly or indirectly controlling the transcription of most 274

flagellar genes. 275

FsnR encodes a C-terminal LuxR-type DNA binding domain and may act as a 276

transcription factor. A recombinant FsnR-His6 protein expressed in E. coli and purified by 277

Ni-NTA affinity chromatography was used in EMSAs to establish whether FsnR binds to the 278

5' promoter of operons Smlt2303 and Smlt2318. The promoter region of the Smlt2303 operon 279

was selected because fsnR is putatively located within this operon. If FsnR binds to the 280

cis-regulatory elements of the Smlt2303 operon, it will form an autoregulation loop. 281

Conversely, the 5' promoter sequence of the Smlt2318 operon was selected because the operon 282

contains 12 genes (Smlt2307-Smlt2318) and is the largest gene cluster of all 283


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flagellar-associated operons. FsnR-His6 formed stable protein-DNA complexes with the 284

promoter sequences of both Smlt2303 (Fig. 6B) and Smlt2318 (Fig. 6C). The addition of 285

unlabeled probes effectively competed with the binding of FsnR-His6 to isotope-labeled 286

probes. These results demonstrated FsnR specifically binds to the promoter regions of the 287

Smlt2303 and Smlt2318 operons and activates their transcription, which may be a critical 288

regulatory process during S. maltophilia biofilm development. 289


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DISCUSSION 290

291

The molecular process of S. maltophilia biofilm formation is important to defend this 292

emergent pathogen (3). Here we identified 46 genes containing mutations associated with 293

changed biofilm formation (Fig. 2 and Table S1). As these genes are involved in multiple 294

biochemical and regulatory cascades, we could surmise that S. maltophilia biofilm formation 295

is a tightly controlled, and highly complex process. With the exception of a few genes, such as 296

xanA which is involved in polysaccharide biosynthesis (12), all remaining genes had not 297

previously been reported, hence their roles in biofilm development require further 298

investigation. We provided evidence that an orphan response regulator, FsnR, directly binds to 299

promoters of at least two flagella-associated operons, activating their transcription (Fig. 6). 300

Inactivation of fsnR resulted in deficiencies in flagella assembly, swimming motility and 301

biofilm formation (Fig. 4 and Fig. 5). These results identify FsnR as a pivotal modulator of S. 302

maltophilia biofilm formation, and as such a desirable target in the development of novel 303

antibacterial agents. 304

In the current study, we identified 17 structural and three regulatory genes associated with 305

flagellar assembly (Table S1). The vital role bacterial flagella play in biofilm development is 306

well recognized (24). Flagella not only contribute to cell motility during biofilm formation 307

and dispersal, but also take part in surface sensing and colonization (25). Consequently, subtle 308

regulation of flagellar biosynthesis and cell motility behavior are important in generating and 309

maintaining the bacterial biofilm matrix. In general, regulatory genes of flagella can be 310

assigned to one of three groups: Group I regulates flagella gene expression and assembly, 311

Group II regulates flagella-associated chemotaxis, and Group III regulates modes of flagella 312


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operation (24). To date, these regulatory genes have not been studied in S. maltophilia. Here 313

we identified a number of regulatory genes. fsnR was characterized as a Group I transcription 314

regulator responsible for activation of a number of flagellar genes of S. maltophilia. BlastP 315

sequence alignment revealed FsnR shared the greatest homology with E. coli UvrY (e = 1e-40, 316

identity = 38%, 100% coverage). Inactivation of uvrY caused deficiencies in biofilm 317

formation and virulence for a number of enterobacterial species. A uvrY gene mutation 318

down-regulated type 1 and Pap fimbriae in E. coli (26). Teplitski et al (2006) reported that 319

uvrY (sirA) inactivation caused the repression of the master regulator of flagellar genes, flhDC 320

in Salmonella enterica (27). The present study provides molecular evidence that FsnR is a 321

transcription activator directly binding to the promoter regions of flagellar genes (Fig. 6B). 322

Collectively, these results indicate that a UvrY/FsnR-like response regulator may modulate 323

flagellar gene expression on multiple levels, or that they have experienced functional 324

differentiation of signal rewiring during bacterial evolution. Furthermore, FsnR is an “orphan” 325

response regulator whose cognate histidine kinase remains unknown. Identification of its 326

cognate histidine kinase, using computational methods for prediction of protein-protein 327

interactions together with phosphorylation profiling (28, 29), will improve our understanding 328

of the effects environmental stimuli have on FsnR-mediated flagella assembly. 329

Four other regulatory genes (Smlt2270, Smlt0158, Smlt2297, and Smlt2324) associated 330

with S. maltophilia biofilm formation were identified in the current study. Smlt2270 (fliA) 331

encodes flagella sigma factor FliA (σ28

). The σ28

-containing holoenzyme of S. typhimurium 332

was shown to selectively bind to the -35 region downstream of fliC and anti-sigma factor fliM, 333

activating their transcription at a late stage of flagella assembly (30). Further to this, σ28

was 334

shown to control type III secretion system gene expression in S. typhi (31), suggesting it is a 335


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global regulator connecting different physiological pathways. Regulatory gene Smlt2297 336

resides in the fsnR operon, hence its transcription is also modulated by FsnR autoregulation. 337

Smlt2297 encodes an alternative sigma factor (σ54

), known to regulate flagellar gene 338

expression in a diverse range of bacterial species, including Enterococcus faecalis (32), Vibrio 339

fischeri (33), Pseudomonas aeruginosa (34), Burkholderia cenocepacia (35). Consequently, 340

inactivation of σ28

or σ54

has been shown to result in deficiencies in motility, virulence and 341

biofilm formation. To date, the consensus binding motifs of σ28

and σ54

in most of the 342

aforementioned bacteria and including S. maltophilia remain unclear. 343

As with fsnR, the two regulatory genes, glnL and ravS, encode histidine kinase sensors 344

belonging to two-component signal transduction systems. GlnL (Smlt0158, NRII) is a 345

putative histidine kinase with a predicted transmembrane domain. The presence of a PAS 346

domain at the N-terminal region of GlnL suggests GlnL has the capacity to detect intracellular 347

redox potentials, oxygen, or other stimuli. Not surprisingly, glnL is situated with a putative 348

cognate response regulator gene, glnG (Smlt0159). GlnG (NRI) is a NtrC-family transcription 349

factor containing the receiver AAA, and a helix-turn-helix output domain. Future studies will 350

focus on the GlnL-GlnG regulon and its relationship with S. maltophilia biofilm formation. 351

The fourth regulatory gene identified in the present study was Smlt2324, an ortholog of the 352

ravS gene of X. campestris. In X. campestris, ravS encodes a PAS domain-containing histidine 353

kinase and appears to constitute a “three-component” signal transduction system (RavSAR) 354

with histidine kinase RavA and response regulator RavR (23, 36). The RavSAR system seems 355

to regulate bacterial gene expression by affecting turnover of the second messenger c-di-GMP; 356

conceivable as RavR encodes both a putative diguanylate cyclase (GGDEF) and c-di-GMP 357

phosphodiesterase (EAL) domain (23). RavSAR gene inactivation markedly attenuated the 358


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virulence of X. campestris (23, 36). However, comparatively little is known of the RavSAR 359

regulation, and it would be worthwhile investigating the role of RavS during RavA-RavR 360

phosphotransfer, the nature of the signals detected by RavS and RavA, and establishing how 361

these three proteins constitute a dynamic protein complex during regulation. 362

Gene mutations causing increased biofilm formation have been identified in other 363

bacterial species, including lipopolysaccharide related genes in E. coli (37), a 364

S-ribosylhomocysteinase gene luxS in Listeria monocytogenes (38), and two-component 365

signaling system genes in P. aeruginosa (39). We identified five genes whose mutants 366

exhibited a substantial increase in biofilm formation compared with that of the WT strain (Fig 367

2A and Table S1). Four of these genes, Smlt3804, Smlt4259, Smlt0490 and Smlt0625 encode 368

enzymes, whilst Smlt0416 encodes a protein of unknown function. Insufficient information 369

was obtained in the current study to elucidate a relationship between biofilm formation and 370

the functionality of these genes. For example, Smlt3804 encodes a glyceraldehyde 371

3-phosphate dehydrogenase (GAPDH) which is involved in glycolysis. Studies have 372

suggested a role for GAPDH in bacterial biofilm formation. GAPDH expression was altered 373

in mutants with biofilm deficiencies for Streptococcus suis and S. mutans (40, 41). In S. oralis, 374

cell-surface GAPDH acts as the binding site for periodontopathic bacteria Porphyromonas 375

gingivalis biofilm formation (42, 43). These studies indicated that GAPDH has a role in 376

bacterial biofilm. However, further experimental evidence is needed to elucidate why 377

mutations in the five genes listed above resulted in an increase in S. maltophilia biofilm 378

production. 379


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ACKNLOWEDGEMENTS 380

381

This study was financially supported by the Strategic Priority Research Program of the 382

Chinese Academy of Sciences (Grant No. XDB11040700), the National Basic Research 383

Program of the Ministry of Science and Technology of China (Grant No. 2011CB100700), 384

National Science Foundation of China (Grant Nos. 31370127, 31070081, and 31400071), and 385

China Ocean Mineral Resources R & D Association (Grant No. DY125-15-T-07). 386


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TABLE 1 Bacterial strains and plasmids used in this study 387

388

Strains or plasmids Genotype or description1 Resources

Strains

S. maltophilis ATCC 13637 WT, wild-type strain CGMCC

E. coli strain DH5α Host strain for molecular cloning Lab collection

E. coli strain BL21(DE3) Host strain for protein expression (KanR) Lab collection

SM001 ΔfsnR, in-frame deletion mutant of fsnR This study

SM002 ΔfsnR-fsnR, genetic complementary strain of

SM001 mutant (StrR)

This study

SM003 CK, SM001 containing a blank pHM1 vector

(StrR)

This study

Clones from mutant library EZ::TN transposon mutants of WT strain with

biofilm deficiency (KanR)

Table S1 for details

Plasmids

pK18mobsacB Suicide vector to create mutant by double

cross-over (KanR)

Schafer et al. 1994

pET30a Protein expression vector (KanR) Novagen

pHM1 Broad-host vector for genetic complementation

(SpR)

Lab collection

pHM2299 pHM1::fsnR, genetic complementary vector (StrR) This study

pET2299 pET30a::fsnR, vector expression FsnR (KanR) This study

1. Kan

R: kanamycin resistant; Str

R: streptomycin resistant. 389


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TABLE 2. Primers used in this study. 390

391

Primer

name

Sequence (5ˊ-3ˊ)1 Purpose

SP1 GATAGATTGTCGCACCTGATTG Specific primer 1, For TAIL-PCR

SP2 AAGACGTTTCCCGTTGAATATG Specific primer 2, For TAIL-PCR

SP3 GCAATGTAACATCAGAGATTTTGAG Specific primer 3, For TAIL-PCR

AD1 NTCGASTWTSGWGTT Arbitrary primer, for TAIL-PCR

AD4 TGWGNANCASAGA Arbitrary primer, for TAIL-PCR

AD5 AGWGNAGWANCAWAGG Arbitrary primer, for TAIL-PCR

AD6 CAWCGICNGAIASGAA Arbitrary primer, for TAIL-PCR

AD8 STTGNTASTNCTNTGC Arbitrary primer, for TAIL-PCR

Kan Sense: GCAATCAGGTGCGACAATC

Antisense: AAGTCAGCGTAATGCTCTGC

DNA probe used in Southern

Blotting

IFD2299 Sense-W: GAATTCTTACTCCAGCTCGTGCTG

Antisense-X: ACTAGTAGCAACAAGGAAATCGCC

Sense-Y: ACTAGTGTCCATCAGGACCACGTC

Antisense-Z: AAGCTTGTGCGAGTTCTCATCGTC

For ΔfsnR mutant construction

C2299 Sense: AAGCTTGTGCGAGTTCTCATCGTC

Antisense: GAATTCTTACTCCAGCTCGTGCTG

For ΔfsnR-fsnR construction

P2299 Sense: CGCCATATGCGAGTTCTCATCGTCGAC

Antisense: AAGCTTCTCCAGCTCGTGCTGATG

For FsnR protein expression


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Pb2303 Sense: GTGATTCTCCTGGATCC

Antisense: CTCTTTCAGCGCTGTACCT

For EMSA probe of Smlt2303

Pb2318 Sense: GGCTGCTGTATCGGCGGGGT

Antisense: GGCGCCTCCTGTCGATGG

For EMSA probe of Smlt2318

0706 Sense: GTGCCGTTGCCGGTCGTAC

Antisense: GGACCTGGTTGTTCGAGC

Semi-qRT-PCR analysis of Smlt0706

0710 Sense: CATCAACCGGCCGTGGCC

Antisense: CTACGGTCTGCTGCAGGG


2274 Sense: GTGGGAGCATTCATGGTC

Antisense: GAAATGAAGGAGAGCGAG


2283 Sense: GAGAACAGCATCAGCAGG

Antisense: CTTCACCCAGTCCAAGCAC


2290 Sense: GACCATCACCTTGGCGAG

Antisense: CTGCCGGGCACGGTGCTG


2297 Sense: GACGGTGGACTCGTGCATG

Antisense: AGTACGAACAGGCGCTGG


2303 Sense: GCTGACGTAGCCGTCGAC

Antisense: TGGGACTGCAGACGCGTG


2306 Sense: GAGATCTGCAGGCCGGAG

Antisense: TCCTTCACCAGCCAGCTG


2318 Sense: GTTCTCGCCTGAGGCGTG

Antisense: GTGCTCGATCCGGCGATG



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2319 Sense: CAACACCACGGTGGAGGT

Antisense: CCGGGCAGCGTCACGCTG


2324 Sense: TCGTCTTCCTGCAGGGAG

Antisense: CAGCTTCCATGCCGGCCTG


tmRNA Sense: GGGGGTGCACTGGTTTCG

Antisense: TGGTGGAGGTGGGCGGAAT

Semi-qRT-PCR analysis of tmRNA

1 Standard IUB/IUPAC nucleic acid codes 392


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FIGURE LEGENDS 393

394

FIG 1. Construction and screening of a random transposon insertion mutant library of S. 395

maltophilia. (A) Determination of randomness, and insertional copy numbers of mutants. 396

Twenty-nine randomly chosen colonies of EZ::TN transposon insertional transformants and of 397

wild-type (WT) strain were analyzed by Southern blotting. Total DNA was extracted from the 398

bacterial strains, digested with SmaI, and fractionated in a 1% agarose gel. A PCR product of 399

EZ::TN transposon was labeled with [α-32

P]-dCTP and used as the DNA hybridization probe. 400

M: 1 kb DNA ladder. (B) Pipeline of screening for mutants with biofilm deficiency. The 401

number of mutants obtained after each round of screening is shown. Details of the screens 402

were described in the Materials and Methods section of this report. 403

404

FIG 2. Mapping the insertional sites on the genome of S. maltophilia. (A) Schematic view of 405

the insertional sites on the genome of S. maltophilia, using the complete genome of K279a as 406

a reference. Coding sequences (CDSs) were as per the genomic annotation for K279a. Genes 407

carrying mutations which resulted in a decrease or increase in biofilm formation are depicted 408

in black and red, respectively. (B) Functional classification of the mutated genes. Detailed 409

information on the mutated genes in (A) and (B) is provided in Table S1. 410

411

FIG 3. Transposon insertions in gene clusters associated with flagella or pili assembly. White 412

arrows indicate CDSs and the direction in which they are transcribed. Black arrows indicate 413

genes disrupted by transposon insertions. Black triangles indicate independent EZ::TN 414

transposon insertions. Virtual Institute of Microbial Stress and Survival (VIMSS) operon 415


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predictions are illustrated below. 416

417

FIG 4. Mutations in fsnR substantially reduced biofilm formation. (A) Crystal violet stained 418

biofilm formed by bacterial strains. (B) Quantification of bacterial biofilm formation by 419

optical density (OD590) measurements. Bars represent standard deviations (n = 4). **, P < 0.01, 420

calculated by Student’s t-test. WT: wild-type strain; ΔfsnR: fsnR in-frame deletion mutant 421

SM001; ΔfsnR-fsnR: complementary strain SM002; CK: ΔfsnR containing a blank pHM1 422

vector SM003. (C) Growth curves of bacterial strains in rich NYG medium. Bars represent 423

standard deviations (n = 4). 424

425

FIG 5. fsnR mutation resulted in deficiencies in swimming motility and flagellar development. 426

(A) Swimming motility of bacterial strains. All strains were inoculated into 0.1% NYG agar 427

and grown for 24 hours. WT: wild-type strain; ΔfsnR: fsnR in-frame deletion mutant SM001; 428

ΔfsnR-fsnR: complementary strain SM002; CK: ΔfsnR containing a blank pHM1 vector 429

SM003. (B) fsnR mutation caused abnormalities in flagella assembly. Bacterial morphology 430

was observed with a scanning transmission electron microscope. Magnification 4000×. Black 431

arrows indicate the presence of flagella. 432

433

FIG 6. FsnR controls expression of flagellar genes and directly binds to promoter regions. (A) 434

Semi-quantitative RT-PCR assays of flagella-associated gene transcriptions. RT represents 435

RT-PCR amplification using cDNAs synthesized from total RNA of bacterial strains; −RT 436

denotes the negative control in which reverse transcriptase was absent during cDNA synthesis; 437

amplification using bacterial DNA and cDNA of tmRNA as templates were used as the 438


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positive control and loading control, respectively. WT: wild-type strain; ΔfsnR: fsnR in-frame 439

deletion mutant; ΔfsnR-fsnR: complementary strain. (B) and (C) EMSA detected FsnR 440

directly binds to promoter regions of Smlt2303 (B) and Smlt2318 (C) operons, respectively. 441

Four fmol of [α-32

P]-dCTP-labeled double-stranded DNA corresponding to the Smlt2303 and 442

Smlt2318 promoter regions, respectively, were used in each reaction. Increasing amounts of 443

unlabeled probes were used as competitors (10 – 250×) for binding to 1 μg of FsnR-His6 444

recombinant protein. Black triangle (right) indicates the protein-DNA binding complexes. 445


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REFERENCES 446

447

1. Crossman LC, Gould VC, Dow JM, Vernikos GS, Okazaki A, Sebaihia M, 448

Saunders D, Arrowsmith C, Carver T, Peters N, Adlem E, Kerhornou A, Lord A, 449

Murphy L, Seeger K, Squares R, Rutter S, Quail MA, Rajandream MA, Harris D, 450

Churcher C, Bentley SD, Parkhill J, Thomson NR, Avison MB. 2008. The 451

complete genome, comparative and functional analysis of Stenotrophomonas 452

maltophilia reveals an organism heavily shielded by drug resistance determinants. 453

Genome Biol. 9:R74. 454

2. Ryan RP, Monchy S, Cardinale M, Taghavi S, Crossman L, Avison MB, Berg G, 455

van der Lelie D, Dow JM. 2009. The versatility and adaptation of bacteria from the 456

genus Stenotrophomonas. Nat. Rev. Microbiol. 7:514-525. 457

3. Brooke JS. 2012. Stenotrophomonas maltophilia: an emerging global opportunistic 458

pathogen. Clin. Microbiol. Rev. 25:2-41. 459

4. Looney WJ, Narita M, Muhlemann K. 2009. Stenotrophomonas maltophilia: an 460

emerging opportunist human pathogen. The Lancet Infect. Dis. 9:312-323. 461

5. Brooke JS. 2014. New strategies against Stenotrophomonas maltophilia: a serious 462

worldwide intrinsically drug-resistant opportunistic pathogen. Expert. Rev. Anti. Infect. 463

Ther. 12:1-4. 464

6. Safdar A, Rolston KV. 2007. Stenotrophomonas maltophilia: changing spectrum of a 465

serious bacterial pathogen in patients with cancer. Cli. Infect. Dis. 45:1602-1609. 466

7. Nicodemo AC, Paez JI. 2007. Antimicrobial therapy for Stenotrophomonas 467

maltophilia infections. Eur. J. Cli. Microbiol. Infect. Dis. 26:229-237. 468


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

28

8. Toleman MA, Bennett PM, Bennett DM, Jones RN, Walsh TR. 2007. Global 469

emergence of trimethoprim/sulfamethoxazole resistance in Stenotrophomonas 470

maltophilia mediated by acquisition of sul genes. Emerg. Infect. Dis. 13:559-565. 471

9. Pompilio A, Piccolomini R, Picciani C, D'Antonio D, Savini V, Di Bonaventura G. 472

2008. Factors associated with adherence to and biofilm formation on polystyrene by 473

Stenotrophomonas maltophilia: the role of cell surface hydrophobicity and motility. 474

FEMS Microbiol. Lett. 287:41-47. 475

10. de Oliveira-Garcia D, Dall'Agnol M, Rosales M, Azzuz AC, Alcantara N, 476

Martinez MB, Giron JA. 2003. Fimbriae and adherence of Stenotrophomonas 477

maltophilia to epithelial cells and to abiotic surfaces. Cell. Microbiol. 5:625-636. 478

11. Jucker BA, Harms H, Zehnder AJ. 1996. Adhesion of the positively charged 479

bacterium Stenotrophomonas (Xanthomonas) maltophilia 70401 to glass and Teflon. J. 480

Bacteriol. 178:5472-5479. 481

12. Huang TP, Somers EB, Wong AC. 2006. Differential biofilm formation and motility 482

associated with lipopolysaccharide/exopolysaccharide-coupled biosynthetic genes in 483

Stenotrophomonas maltophilia. J. Bacteriol. 188:3116-3120. 484

13. Ryan RP, Fouhy Y, Garcia BF, Watt SA, Niehaus K, Yang L, Tolker-Nielsen T, 485

Dow JM. 2008. Interspecies signalling via the Stenotrophomonas maltophilia 486

diffusible signal factor influences biofilm formation and polymyxin tolerance in 487

Pseudomonas aeruginosa. Mol. Microbiol. 68:75-86. 488

14. Roscetto E, Angrisano T, Costa V, Casalino M, Forstner KU, Sharma CM, Di 489

Nocera PP, De Gregorio E. 2012. Functional characterization of the RNA chaperone 490

Hfq in the opportunistic human pathogen Stenotrophomonas maltophilia. J. Bacteriol. 491


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

29

194:5864-5874. 492

15. Lin YT, Huang YW, Liou RS, Chang YC, Yang TC. 2014. MacABCsm, an 493

ABC-type tripartite efflux pump of Stenotrophomonas maltophilia involved in drug 494

resistance, oxidative and envelope stress tolerances and biofilm formation. J. 495

Antimicrob. Chemoth. doi: 10.1093/jac/dku317 496

16. Goryshin IY, Jendrisak J, Hoffman LM, Meis R, Reznikoff WS. 2000. Insertional 497

transposon mutagenesis by electroporation of released Tn5 transposition complexes. 498

Nat. Biotechnol. 18:97-100. 499

17. Qian W, Jia Y, Ren SX, He YQ, Feng JX, Lu LF, Sun Q, Ying G, Tang DJ, Tang H, 500

Wu W, Hao P, Wang L, Jiang BL, Zeng S, Gu WY, Lu G, Rong L, Tian Y, Yao Z, 501

Fu G, Chen B, Fang R, Qiang B, Chen Z, Zhao GP, Tang JL, He C. 2005. 502

Comparative and functional genomic analyses of the pathogenicity of phytopathogen 503

Xanthomonas campestris pv. campestris. Genome Res. 15:757-767. 504

18. Sun Q, Wu W, Qian W, Hu J, Fang R, He C. 2003. High-quality mutant libraries of 505

Xanthomonas oryzae pv. oryzae and X. campestris pv. campestris generated by an 506

efficient transposon mutagenesis system. FEMS Microbiol. Lett. 226:145-150. 507

19. Sambrook JR, D. W. 2001. Molecular Cloning. Cold Spring harbor Laboratory, Cold 508

Spring Harbor, New York. 509

20. Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A. 1994. Small 510

mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids 511

pK18 and pK19: selection of defined deletions in the chromosome of 512

Corynebacterium glutamicum. Gene 145:69-73. 513

21. Musken M, Di Fiore S, Romling U, Haussler S. 2010. A 96-well-plate-based optical 514


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

30

method for the quantitative and qualitative evaluation of Pseudomonas aeruginosa 515

biofilm formation and its application to susceptibility testing. Nat. Protoc. 516

5:1460-1469. 517

22. O'Toole GA, Kolter R. 1998. Initiation of biofilm formation in Pseudomonas 518

fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic 519

analysis. Mol. Microbiol. 28:449-461. 520

23. He YW, Boon C, Zhou L, Zhang LH. 2009. Co-regulation of Xanthomonas 521

campestris virulence by quorum sensing and a novel two-component regulatory 522

system RavS/RavR. Mol. Microbiol. 71:1464-1476. 523

24. Guttenplan SB, Kearns DB. 2013. Regulation of flagellar motility during biofilm 524

formation. FEMS Microbiol. Rev. 37:849-871. 525

25. Belas R. 2014. Biofilms, flagella, and mechanosensing of surfaces by bacteria. Trends 526

in Microbiol.. 22:517-527. 527

26. Mitra A, Palaniyandi S, Herren CD, Zhu X, Mukhopadhyay S. 2013. Pleiotropic 528

roles of uvrY on biofilm formation, motility and virulence in uropathogenic 529

Escherichia coli CFT073. PloS One 8:e55492. 530

27. Teplitski M, Al-Agely A, Ahmer BM. 2006. Contribution of the SirA regulon to 531

biofilm formation in Salmonella enterica serovar Typhimurium. Microbiol. 532

152:3411-3424. 533

28. Skerker JM, Perchuk BS, Siryaporn A, Lubin EA, Ashenberg O, Goulian M, 534

Laub MT. 2008. Rewiring the specificity of two-component signal transduction 535

systems. Cell 133:1043-1054. 536

29. Skerker JM, Prasol MS, Perchuk BS, Biondi EG, Laub MT. 2005. Two-component 537


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

31

signal transduction pathways regulating growth and cell cycle progression in a 538

bacterium: a system-level analysis. PLoS Biol. 3:e334. 539

30. Schaubach OL, Dombroski AJ. 1999. Transcription initiation at the flagellin 540

promoter by RNA polymerase carrying sigma28 from Salmonella typhimurium. J. Biol. 541

Chem. 274:8757-8763. 542

31. Eichelberg K, Galan JE. 2000. The flagellar sigma factor FliA (sigma(28)) regulates 543

the expression of Salmonella genes associated with the centisome 63 type III secretion 544

system. Infect. Immun. 68:2735-2743. 545

32. Iyer VS, Hancock LE. 2012. Deletion of sigma(54) (rpoN) alters the rate of autolysis 546

and biofilm formation in Enterococcus faecalis. J. Bacteriol. 194:368-375. 547

33. Wolfe AJ, Millikan DS, Campbell JM, Visick KL. 2004. Vibrio fischeri sigma54 548

controls motility, biofilm formation, luminescence, and colonization. Appl. Environ. 549

Microbiol. 70:2520-2524. 550

34. Thompson LS, Webb JS, Rice SA, Kjelleberg S. 2003. The alternative sigma factor 551

RpoN regulates the quorum sensing gene rhlI in Pseudomonas aeruginosa. FEMS 552

Microbiol. Lett. 220:187-195. 553

35. Saldias MS, Lamothe J, Wu R, Valvano MA. 2008. Burkholderia cenocepacia 554

requires the RpoN sigma factor for biofilm formation and intracellular trafficking 555

within macrophages. Infect. Immun. 76:1059-1067. 556

36. Tao J, Li C, Luo C, He C. 2014. RavA/RavR two-component system regulates 557

Xanthomonas campestris pathogenesis and c-di-GMP turnover. FEMS Microbiol. Lett. 558

358:81-90. 559

37. Nakao R, Ramstedt M, Wai SN, Uhlin BE. 2012. Enhanced biofilm formation by 560


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

32

Escherichia coli LPS mutants defective in Hep biosynthesis. PLoS One 7:e51241. 561

38. Sela S, Frank S, Belausov E, Pinto R. 2006. A Mutation in the luxS gene influences 562

Listeria monocytogenes biofilm formation. Appl. Environ. Microbiol. 72:5653-5658. 563

39. Zhang L, Fritsch M, Hammond L, Landreville R, Slatculescu C, Colavita A, Mah 564

TF. 2013. Identification of genes involved in Pseudomonas aeruginosa 565

biofilm-specific resistance to antibiotics. PLoS One 8:e61625. 566

40. Palmer SR, Crowley PJ, Oli MW, Ruelf MA, Michalek SM, Brady LJ. 2012. 567

YidC1 and YidC2 are functionally distinct proteins involved in protein secretion, 568

biofilm formation and cariogenicity of Streptococcus mutans. Microbiol. 569

158:1702-1712. 570

41. Wang Y, Zhang W, Wu Z, Zhu X, Lu C. 2011. Functional analysis of luxS in 571

Streptococcus suis reveals a key role in biofilm formation and virulence. Vet. 572

Microbiol. 152:151-160. 573

42. Maeda K, Nagata H, Kuboniwa M, Ojima M, Osaki T, Minamino N, Amano A. 574

2013. Identification and characterization of Porphyromonas gingivalis client proteins 575

that bind to Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase. Infect. 576

Immun. 81:753-763. 577

43. Nagata H, Iwasaki M, Maeda K, Kuboniwa M, Hashino E, Toe M, Minamino N, 578

Kuwahara H, Shizukuishi S. 2009. Identification of the binding domain of 579

Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase for Porphyromonas 580

gingivalis major fimbriae. Infect. Immun. 77:5130-5138. 581


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1 2 3 4 5 6 7 8 9 10 11 12 13 14

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 WT M

1 kb

2 kb

3 kb

4 kb

6 kb

10 kb

1 kb

2 kb

3 kb

4 kb

6 kb

10 kbEZ::TN transposon mutant library

(14,208 clones)

396 primary positive mutants

Primary screen

Progeny production

99 positive mutants

Test for auxotrophy and slow growth

Four repeated experiments

53 auxotrphy or slow growth

19 with increased biofilm 80 with decreased biofilm

6 unique sequences 68 unique sequences

13 Incorrect sequences 12 Incorrect sequences

46 genes

4 intergenic regions

2 undefined regions

A B

152 positive mutants

244 false positive clones

Fig. 1


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A

4,851,126 bp

1,2

12,7

80 b

p

2,425,563 bp

3,6

38,3

40 b

p

Sm

lt0158

Sm

lt0208

Sm

lt0209

Sm

lt0276

Sm

lt04

16

Sm

lt04

17-0

418

Smlt0

490

Smlt0518

Smlt0625

Smlt0639

Smlt0653

Smlt0684

Smlt0707

Smlt0708

Smlt0709

Smlt0845Smlt0857

Stenotrophomonas maltophiliausing K279a complete genome as a reference

Smlt1613Smlt1614

Smlt2272

Smlt2072Sm

lt2273

Smlt2280

Smlt2281

Sm

lt2282

Sm

lt2284

Sm

lt2286

Sm

lt2289

Sm

lt2297

Sm

lt2299

Sm

lt2303

Sm

lt2308

Sm

lt2310

Sm

lt2311

Sm

lt2312

Sm

lt2314

Sm

lt2324

Smlt3167Smlt3

426

Smlt3804

Sm

lt4318

Sm

lt4552

Sm

lt4645-4

647

Smlt0722-0723

Sm

lt4554

Sm

lt4259

Smlt0704-0706

Sm

lt04

18

Smlt3195

Smlt2070

B

Flagella and pili structural genes

Polysaccharide and carbohydrate

Purine biosynthesis

Transpotation

Gene regulation

Peptide and lipid synthesis

Oxioreduction

Function unknown

Transposable elements

Intergenic region

Undefined

17

4

63

5

3

3

4

1

42

Fig. 2


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2270 2271 2272 2273 2274 2275 2276 2277 2278 2279 2280 2281 2282 2283 2284 2285 2286 2287 2288 2289 2290 2291 2292Smlt2263

fliA flhF flhA flhB fliR fliQ fliP fliO fliN fliM fliL fliK fliJ fliI fliH fliG fliF fliE

2297

ISPsy9 ISPsy9

Smlt2293 2294 2295 2296 2299 2300 2301 2302 2303 2304 2305 2306 2307 2308 2309 2310 2311 2312 2313 2314 2315 2316 2317

NRI SigmaN fliS fliD fliC flaA flgI flgH flgG flgF flgD flgC flgB

Smlt2319 2320 2321 2322 2323 2324 2325 2326

flgA flgM flgN ravA ravR ravSSmlt0706 0707 0708 0709 0710 0711 0712 0713

smf-1 mrkC wecB nfrB

Putative operon

Putative operon Putative operon Putative operon

Putative operon Putative operon Putative operon Putative operon

2318

Putative operon Putative operon

......

fsnR flgL flgK flgJ flgE

Fig. 3


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WT ΔfsnR ΔfsnR-fsnR CK

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

WT ΔfsnR ΔfsnR-fsnR CK

AB

S O

D590

** **

B

C

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

10 20 30

AB

S O

D600

Time (h)

WT

ΔfsnR

ΔfsnR-fsnR

CK

A

Biofilm

Fig. 4


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WT ΔfsnR

ΔfsnR-fsnRCK

WT

(Φ = 3.13 ± 0.09 cm)

ΔfsnR

(Φ = 0.45 ± 0.07 cm)

CK

(Φ = 0.48 ± 0.04 cm)

ΔfsnR-fsnR

(Φ = 1.58 ± 0.10 cm)

A B

Fig. 5


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tmRNA

Smlt0706

Smlt0710

Smlt2274

Smlt2283

Smlt2290

Smlt2297

Smlt2303

Smlt2318

Smlt2319

Smlt2324

Smlt2306

-RT

WT DNAΔfsnR-

fsnR

Probe: 5’ region of Smlt2303

Free

probe FsnR 10× 50× 200×

Probe: 5’ region of Smlt2318

Free

probeFsnR 25× 250×100×

A B

C200×

ΔfsnR CK

Fig. 6


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aem accepts, published online ahead of print on 5 december ...€¦ · 02/12/2014 · 5 83...

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